Gap junctions (GJs) connect the interiors of neighboring cells and permit the passive cell-to-cell flux of molecules and ions less than approximately 1 kDa. GJs have the potential to modify conventional and non-conventional cancer therapies in several ways, but this has seen little investigation. I propose to investigate how GJs alter the cytotoxicity of chemotherapeutic agents known as ribonucleotide reductase (RR) inhibitors. These agents induce nucleotide imbalances in cells, and I hypothesize nucleotide buffering via GJs will impact RR toxicity. I have proposed two Specific Aims to address the hypothesis: Aim 1: Isolate clones of GJ-competent and GJ-incompetent WB cells that are sensitive and resistant to the RR inhibitor, hydroxyurea (HU). We will use WB-F344 (GJ+) and WB-aB1 (GJ-) cells for this purpose and will characterize HU cytotoxic dose response, nucleotide pools, and RR activity and expression. Aim 2: Quantify the effects of GJs on cytotoxic response and nucleotide buffering in co-cultures of HU-sensitive and resistant cells. HU-sensitive and resistant, GJ+ or GJ- cells will be co-cultured and treated with HU, then cytotoxicity and nucleotides will be quantified in each type of cell. These studies may lead to new cancer therapy paradigms and treatments.